Self-assembly of proteins and peptides into nanoaggregates of various sizes and morphologies is a widespread phenomenon reported for a large number of proteins and synthetic peptides. Misfolding and aggregation of proteins are associated with a wide range of human pathologies termed protein misfolding or deposition neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. Despite the important contribution of protein misfolding and spontaneous aggregation to disease pathology, very little is known about the molecular mechanisms underlying these processes. It has been shown that oligomeric species rather long filaments are neurotoxic, but the nature of these species remains unknown. The objective of this application is to characterize the nano-oligomers formed at the early stages of self-assembly and identify the species critically involved in aggregation. The oligomers are formed transiently during the aggregation process, and their characterization requires special approaches capable of probing such species. This application proposes a set of such approaches to attain our objective. Based on preliminary data, the central hypothesis is that formation of dimers is the key step for promoting aggregation and that the lifetimes of dimeric complexes define the misfolding-aggregation paths. The rationale for the proposed research is that understanding fundamental mechanisms of protein misfolding and aggregation has the potential to translate into specific approaches to control the aggregation process. These advances will lead to the development of new and innovative preventions and treatments of protein misfolding diseases. Guided by strong preliminary data, our major hypothesis will be tested by pursuing three specific aims: 1) Identify key nuclei for the aggregation kinetic;2) Image directly early stages of the self-assembly process;and 3) Develop novel linkers and tethers for AFM experiments. Under the first aim, the characterization of oligomeric states will be performed. A novel nanoapproach capable of probing transiently formed oligomeric species will be developed. Under the second aim, the assembly of oligomers of each size starting from dimers will be visualized directly. We will be able to measure directly the time-dependent change of the concentration of each oligomer and to identify which species play a role of building blocks for the growth of aggregates. It is hypothesized that the fibrilization requires formation of oligomers of a defined size. We will be able to directly test this hypothesis. A novel nanoimaging approach capable of unambiguous visualizing each oligomer according to its size and quantitative analysis of the kinetics of their formation will be developed. Under the third aim, a novel type of polymer linker will be developed to facilitate completion of the previous aims. The approach is innovative, because it presents a novel model for the protein aggregation process and develops a set of novel nanotechnology methods with broad biomedical applications to test this model. The proposed research is significant because the findings will lay the foundation for developing efficient treatments of protein misfolding diseases at the very early stages. PUBLIC HEALTH RELEVANCE: The work proposed is relevant to public health because the discovery of critical species triggering protein aggregation is expected to increase the understanding of molecular mechanisms of Alzheimer's, Parkinson's and other protein misfolding diseases. The proposed research will provide knowledge on how pathologies related to protein misfolding develop as well as how to control the aggregation process. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will improve preventive and therapeutic treatments for diseases.